Heat stress leads to decreased maize yield (Peters et al., 1971; Thompson, 1975; Chang, 1981; Christy and Williamson, 1985). This can be attributed to reduced photosynthate availability and transportation from source to sink tissues, poor pollination, reduced cell and granule size and number, early seed abortion and/or reduced grain filling period. Growth of endosperm starts with a lag phase in which cells actively divide and continues with a linear phase in which cells increase in size and starch synthesis occurs. Elevated temperature during lag phase resulted in reduced yield (Jones et al., 1984). These investigators suggested that reduced yield was due to reduced cell and granule number and size as well as seed abortion. Additionally, elevated temperatures during the linear phase resulted in shorter grain filling period and subsequently smaller kernels (Jones et al., 1984). Similar results were found by Hunter et al. (1977) and Tollenaar and Bruulsema (1988).
Records from five states that traditionally produce more than 50% of the US corn showed that average daily temperature was 23.6° C., around 2° C. higher than optimum during grain filling (Singletary et al., 1994). Photosynthate availability during grain filling is not reduced at high temperatures, at least in barley and wheat. Indeed, sucrose content in barley and wheat seeds was either unchanged or elevated at high temperatures (Bhullar and Jenner, 1986; Wallwork et al., 1998). Also photosynthesis in maize increases up to 32° C. (Duncan and Hesketh, 1968; Hofstra and Hesketh, 1969; Christy et al., 1985). Moreover, Cheikhn and Jones (1995) studied the ability of maize kernels to fix 14C sucrose and hexoses at different temperatures. They found that these sugars increased in the seed at elevated-temperatures. The evidence above suggests that limited sugar availability and transport into the kernel during grain filling are not the cause of temperature-induced yield decreases.
There have been extensive efforts to identify biochemical pathways that impact grain filling during elevated temperatures. Singletary et al. (1993; 1994) assayed starch biosynthetic enzymes in maize kernels grown in vitro at elevated temperatures (22° C. to 36° C.). They found that ADP-glucose pyrophosphorylase (AGPase) and soluble starch synthase (SSS) were more heat labile compared to other enzymes participating in starch synthesis. They suggested that heat lability of AGPase and SSS contributes to grain filling cessation. Duke and Doehlert (1996) found that transcripts of several genes encoding enzymes of the starch synthesis pathway, including those encoding AGPase, were decreased at 35° C. compared to 25° C. However, enzyme assays showed that only AGPase activity was strikingly lower. They suggested that this could be due to a higher turnover rate of AGPase compared to other enzymes. Finally, Wilhelm et al. (1999), through Q10 analysis, showed that AGPase had the most pronounced reduction in activity compared to several other enzymes. Maize AGPase indeed lost 96% of its activity when heated at 57° C. for 5 min (Hannah et al., 1980).
AGPase catalyzes the first committed step in starch (plants) and glycogen (bacteria) synthesis. It involves the conversion of glucose-1-P (G-1-P) and ATP to ADP-glucose and pyrophosphate (PPi). AGPase is a heterotetramer in plants consisting of two identical small and two identical large subunits. The large and the small subunits are encoded by shrunken-2 (Sh2) and brittle-2 (Bt2) respectively in maize endosperm. AGPase is allosterically regulated by small effector molecules that are indicative of the energy status of the cell. AGPase is activated by 3-PGA, the first carbon assimilatory product, and inhibited/deactivated by inorganic phosphate (Pi) in cyanobacteria, green algae and angiosperms.
The importance of maize endosperm AGPase in starch synthesis has been shown by the kernel phenotype of mutants in either subunit of the enzyme. Indeed, such mutants result in shrunken kernels and a large reduction in endosperm starch content (Tsai and Nelson, 1966; Hannah and Nelson, 1976). There is also evidence that AGPase catalyses a rate-limiting step in starch synthesis (Stark et al. 1992; Giroux et al. 1996; Greene et al. 1998; Sakulsingharoja et al. 2004; Obana et al. 2006; Wang et al. 2007).
Greene and Hannah (1998a) isolated a mutant form of maize AGPase with a single amino acid change in the large subunit termed HS33. They showed that the altered enzyme was more heat-stable and that stability was due to stronger subunit-subunit interactions. When wheat and rice were transformed with a Sh2 variant that contains the HS33 change along with a change that affects the allosteric properties of AGPase (Rev6) (Giroux et al., 1996), yield was increased by 38% and 23% respectively (Smidansky et al., 2002; 2003). Remarkably, the increase was due to an increase in seed number rather than individual seed weight.
Transformation of maize with the Sh2 variant containing the Rev6 and HS33 changes also gives rise to enhanced seed number. Seed yield/ear can be increased up to 68% in maize. A detailed characterization of the maize transgenic events is under way (Greene and Hannah, in preparation). Enhanced seed number cannot be explained by Rev6 since, when expressed alone in maize, it increases only seed weight (Hannah, unpublished). The above studies show the importance of AGPase heat stability in cereal yield.
Cross et al. (2004) generated a mosaic small subunit (MP) consisting of the first 200 amino acids of BT2 and the last 275 amino acids of the potato tuber small subunit. MP in a complex with SH2 had several features that could lead to agronomic gain (Cross et al., 2004; Boehlein et al., 2005). Some of those features were increased activity in the absence of the activator 3-PGA, increased affinity for 3-PGA and elevated heat stability compared to wildtype maize endosperm AGPase (BT2/SH2). Preliminary data show that maize plants with transgenic MP containing AGPase variant expressed in maize endosperm provides for a starch yield increase (Hannah, unpublished data).